The objectives of this proposal are to gain a complete understanding of the structure-function relationships and molecular pathology of the glycoprotein (GHP) Ib-IX-V complex, a platelet membrane receptor essential for normal hemostasis and possibly involved in the development of pathological thrombosis. GP Ib-IX-V exerts its function by binding an adhesive molecule of the subendothelium and plasma, von Willebrand factor (vWF), and the platelet agonist alpha-thrombin. These interactions establish a unique role for the GP Ib-IX-V receptor in initiating platelet adhesions at sites of vascular injury and in supporting platelet aggregation under conditions of high shear stress. GP Ib, GP IX and GP V are distinct proteins that form a noncovalent hetero-oligomeric complex in the platelet membrane; GP Ib itself is composed of one alpha- and one Beta-chain, each the product of a distinct gene, covalently linked through interchain disulfide bonds. Assembly of the complex (at least GP Ib and GP IX) is necessary for efficient platelet-surface expression and complete functional integrity, but the ligand specificity of the complex, that is, the ability to interact with vWF and alpha-thrombin, is an exclusive attribute of the alpha-chain of GP Ib. The studies proposed in this application are based on an integrated approach involving the use of recombinantly-derived fragments, selected monoclonal antibodies, expression in heterologous cells and in genetically-engineered platelets to define the molecular bases of the vWF-binding and alpha-thrombin-binding functions of this receptor complex. Specifically, studies characterizing the structural and functional properties of a recombinant fragment corresponding to the amino-terminus of the GP Ibalpha-chain will identify the essential elements that generate the ligand binding sites for vWF and alpha- thrombin (Aims 1 and 2). Heterologous cell expression will identify the structural elements of the GP Ib-IX complex that support intracellular assembly and surface-expression, with particular regard to the role of the leucine-rich repeats present in each of the components of the complex (Aim 3). In human pathology, a defective GP Ib-IX-V receptor leads to the congenital bleeding disorder known as Bernard-Soulier syndrome. Studies will be devoted to the definition of molecular defects responsible for this disease, with special emphasis given to variants expressing a dysfunctional GP Ib-IX-V complex (Aim 4). Finally, the experiments proposed in Aim 5 will evaluate hypotheses generated with the experiments of Aims 1-4 by taking advantage of an in vivo model of the Bernard-Soulier syndrome, created by mutagenesis of the mouse GP Ibalpha gene, that will permit genetic manipulation of the alpha-subunit of GP Ib via transgenic expression. Definite knowledge will, thus, be obtained on the effects of shear forces on platelet function, the modulation of interaction with vWF, and the signal transducing properties of the receptor. These studies will provide relevant information for (i) understanding the pathogenesis of the thrombotic complications of atherosclerosis, (ii) unraveling the physiological mechanisms of platelet adhesion and activation, (iii) elucidating the molecular pathology of different forms of the Bernard-Soulier syndrome, and (iv) helping in the design of new anti-thrombotic strategies.